Low-dose antigenic signals to helper lymphocytes

CELLULAR I]VI~fUNOLOGY 28, 125--132

(1977)

Low-Dose Antigenic Signals to Helper Lymphocytes1 ~V~ARILYN BALTZ 2 AND ~V~ARVIN ]~. RITTENBERG 3

Department o] Microbiology and Immunology, University o] Oregon Health Sciences Center, Portland, Oregon 97201 Received September 21, 1976 Ealb/c spleen cells from keyhole limpet hemocyanin ( K L H ) primed mice exposed to picogram quantities of K L H for 15 rain in vitro became susceptible to inactivation by antiimmunoglobulin and complement. Susceptibility to antiimmunoglobulin and complement was detected as the loss of helper funo_tion measured by the inability of such cells to cooperate in an in vitro antihapten response to trinitrophenyl-KLH. Helper cells to chicken gamma globulin in the same mixture were not affected indicating specificity of the effect. The induction of susceptibility was temperature dependent; it could be induced at 37 ° or 30 ° but not at 4 ° . Cells became maximally susceptible to antiimmunoglobulin and complement within 15 min after exposure to the low dose of carrier after which susceptibility was lost indicating that the induced state was transient. A model is proposed for low dose dependent arming of helper cells which would permit them to discriminate between antigen challenge and background noise.

INTRODUCTION Lymphocytes are subject to many types of control administered by other cells or cell products (1-3) and antigens (4-6). In addition lymphocytes may need intrinsic control(s) in order to respond to these external influences. While it is possible that a particular matrix array of antigenic determinants is necessary to obtain the signal for triggering (5), it may not be sufficient, and a second, inducing (mitogenic) impulse may also be necessary (7). If both antigenic and mitogenic signals are essential, a required coincidence in the timing of these impulses could provide one form of control to distinguish noise from real events. If, as has been suggested, only a mitogenic signal is required (8), then some means of distinguishing between true signals and random hits appears essential. The signal studies discussed above have referred to B cell triggering; although the receptors on T cells may be different (9), the activation processes presumably would respond to similar control mechanisms. We have made a preliminary observation (10) that we believe bears on the question of discriminatory thresholds in helper cells and provides a 1 Supported by N I H Grant CA 12355 and the Oregon Heart Association and by fellowships from the National Foundation March of Dimes and the American Association of University Women. 2 Present Address: Tumor Immunology Unit Zoology Department, University College London, Gower Street, London, W C 1E 6BT, GB. 3Address reprint requests to M. B. Rittenberg, Ph.D., Department of Microbiology and Immunology, University of Oregon Health Sciences Center, 3818 S. W. Sam Jackson Park Road, Portland, Oregon 97201. 125 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

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model which could apply to all forms of signal hypothesis for triggering immune lymphocytes. T cells appear to recognize antigen via a specific receptor on the cell surface although the nature of the receptor is still controversial (9). Furthermore, the sequence of events after antigen recognition is unknown as indicated above due in part to the small number of specific immune cells in a normal population. Signals delivered to such a small number of cells generally must be detected indirectly and usually require an amplification system; functional analysis can serve this purpose. We have used the helper function as the amplifier to show that subimmunogenic doses (picograms) of carrier can induce specific helper cells to become susceptible to inactivation by antiimmunoglobulin (anti-Ig) and complement (C). This susceptibility is shown to be transient and temperature dependent. Based on these findings we propose a regulatory mechanism which would permit a helper cell to discriminate between background noise and true antigenic insult. MATERIALS

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METHODS

Animals. Female or male Balb/c mice were obtained from Simonsen Laboratories Inc., Gilroy, California, or Texas Inbred Laboratories, Houston, Texas. They were used when 2 to 6 months old. Antigens. Hemocyanin ( K L H ) was obtained from keyhole limpets (Megathura crenulata) by the method of Campbell et al. (11). Trinitrophenylated-KLtt ( T N P K L H ) was prepared by the method of Rittenberg and Amkraut (12) and had an approximate molar ratio of TNPlo00KLH. Dinitrophenylated chicken gamma globulin (DNP6.6-CGG) and CGG were gifts from Dr. N. A. Mitchison. Immuniaations. Particulate K L H or CGG was prepared by coating the protein onto bentonite according to the method of Gallily and Garvey (13) as described previously for T N P - K L H (14). Mice were given intraperitoneal injections of protein-bentonite once a week for 3 consecutive weeks. Each injection contained 100/~g protein in 0.5 ml saline. Cell culture. Mouse spleen cells were cultured by a micromodification (15) of the method of Mishell and Dutton (16). Culture medium was supplemented with 5 × 10-5 M 2-mercaptoethanol (17). TNP-Plaque Assay. Cells synthesizing anti-TNP or anti-DNP antibody were detected by plaque assay using TNP-conjugated sheep ertyhrocytes (TNP-SRBC) (14) and the plaque assay technique of Cunningham and Szenberg (18). C was absorbed with SRBC prior to use (19). Complement. Guinea pig serum (Rockland, Gilbertsville, Pennsylvania) absorbed with agarose (20) was used as a source of C in cell depletion experiments at 1 : 6 final dilution. Rabbit antimouse brain-associated antiserum (anti-T). Rabbit anti-T antiserum was prepared according to the method of Golub (21). Pooled serum was heat inactivated at 56 ° for 30 rain and absorbed with an equal volume of Balb/c liver. At 1 : 10 dilution plus C the absorbed antiserum killed 15% of Balb/c spleen cells, 90% of Balb/c thymus cells and depleted primary in vitro anti-SRBC and anti-TNP-KLtt responses 100% and 97% respectively. Under the same conditions the antiserum had no effect on the in vitro anti-TNP response to TNP-T4, a thymus independent antigen (22). Absorption with Balb/c brain removed all cytotoxic activity. T cells were depleted using the two step procedure of Chan et a,1. (23).

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Rabbit antimouse 19 antisera. Three antisera were used in these experiments. Number 1 (Fig. 2) made against MOPC-195 (IgGxbk) was a gift from Dr. E. Rabellino and was absorbed with 5 × 107 Balb/c thymocytes per ml before use; at 1 : 5 dilution it killed 11% of thymocytes and 21% of spleen cells. Number 2 (Fig. 3) was prepared against purified mouse IgG (Pentex, Miles Laboratories, Kankakee, I L ) . A 1 : 5 dilution killed 10% of thymocytes and 33% of spleen cells. Number 3 (Fig. 4) was prepared against goat antimouse Ig-mouse Ig complexes formed at equivalence. At 1 : 75 dilution it killed 1 5 ~ of thymus cells and 32% of spleen cells. All sera were heat inactivated before use. Antirabbit It7 facilitating or enhancing antiserum. A complement-fixing goat antimouse Ig antiserum (14) which cross-reacted with rabbit Ig but which was not cytotoxic for mouse spleen cells or thymus cells was used at 1 : 10 dilution. The antiserum was heat inactivated and absorbed 3 times with SRBC before use. This antiserum could not substitute for the antimouse Ig antisera described above. Statistical analysis. Data were analyzed by two way analysis of variance; an F test was used for significance testing (24). Experimental Design. The specific experimental protocol is outlined in Fig. 1. Mice were primed with either I
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FIG. I. Experimental design used to demonstrate that low doses of carrier can induce helper cells susceptibility to anti-Ig and C.

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CGG-primed spleen cells were used where indicated. Spleen cells were incubated with diluent only (control cells) or with varying doses of K L H for 60 rain at 4 ° (signal step). Cells were centrifuged through fetal calf serum and washed in the cold to remove unbound K L H and incubated for 15 rain at either 30 ° or 37 ° to allow for any membrane movement or metabolism necessary for signal translation (induction). The temperature was lowered to 4 ° and rabbit antimouse Ig antiserum was added and allowed to bind at 4 ° for 60 rain (direct technique). Cells were washed free of unbound anti-Ig. In some experiments the effect of the rabbit antimouse Ig was amplified by adding antirabbit Ig antiserum (indirect technique) before adding C. Presumably as in the enhancement of IgG plaques (25) this step would facilitate C binding and enhance cell killing. If an amplifying serum was used, it was allowed to bind at 4 ° for 60 min and the cells were then washed. C was added and the cells were incubated at 37 ° for 45 rain and then washed extensively. B cells inactivated by the anti-Ig and C treatment were replenished by adding anti-T and C treated nonprimed spleen cells or intact nonprimed spleen cells. Cells were then cultured with optimal doses of antigen ( T N P - K L H or DNP-CGG) or with no antigen. Direct antihapten PFC were measured on Day 5 of culture. Background (no antigen) plaques were subtracted from all responses.

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FIG. 2. Ability of low doses of KLI-I to induce helper cell susceptibility to anti-Ig and C. Each point represents the mean PFC/106 cells assayed of triplicate cultures. KLH-primed spleen cells were signaled with K L H (10 -B # g to 10 -s ~ g ) or with diluent (control cells) and induced at 37 ° for 15 rain. Control and signaled cells were treated with anti-Ig and C by the indirect technique. Normal spleen cells were used to replenish 13 ceils. 0.02 /~g T N P - K L I - I was used in culture and cells were assayed on Day 5 of culture against T N P - S R B C . Responses of cells signaled with KLI-I differed significantly ( P < 0.001) when tested as a unit against controls.

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with low doses of KLH. A 1 : 1 mixture of CGG and KLH primed spleen cells was signaled with IKLH prior to anti-Ig and C treatment (indirect technique). Normal spleen cells treated with anti-T antiserum and C were used to replenish B cells. Cells were cultured without antigen or with 0.2 /~g DNP-CGG or 0.02 /xg TNP-KLH. Cells were assayed against TNPSRBC on day 5 of culture. Results are expressed as % of control response with control cells treated as in Figure 2. Each point represents a pool of 8 replicate microcultures. RESULTS

Low Doses os Carrier fnd'uee Helper Cell Susceptibility to Anti-Iy a1~d C Figure 2 shows representative data from experiments which suggested that very low signal doses of K L H were effective in inducing K L H helper cell susceptibility to anti-Ig and C. Cells signaled with 10-6 t~g, 10-7 #g, or 10 -8 vg K L H and treated by the indirect anti-Ig technique generated responses equal to 50%, 27% and 59% respectively of the control responses (cells signaled with diluent only prior to anti-Ig and C treatment). The response of signaled cells differed significantly ( P < 0.001) when tested as a unit against the control. In this experiment the 10 -z t*g K L H signal was most effective as an inducer of susceptibility to anti-Ig and C; however, in other experiments 10 -0 #g or 10 -s tzg have sometimes been more effective. The relative heterogeneity of the helper cell population at the time of signal may account for the dose variation as well as for the inability to achieve 100% depletion of helper function.

Specificity os Induction W e used a I : 1 mixture of K L H - p r i m e d spleen cells and CGG-primed spleen cells to determine the specificity of induction by K L H . In the experiment shown in Fig. 3 cells were signaled with diluent (control cells) or with 10-5 tzg to 10-z ~g K L H . All cells were incubated at 37 ° for 15 rain after signaling and treated with anti-Ig and C (indirect technique). Anti-T and C treated normal spleen cells were used to replenish B cells prior to culture without antigen or with optimal doses of T N P - K L H or D N P - C G G . Only KLH-specific helper cells were depleted as indicated by the data which show that the a n t i - D N P response to D N P - C G G

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remained at or above control values, while the anti-TNP response to T N P - K L H was reduced when 10-0 ~ag or 10-~ vg K L H was used as inducer. This experiment suggested carrier specificity in the induction process and argued against a nonspecific effect either on B cells or on adherent cells since the latter are required for in vitro responses to both DNP-CGG and T N P - K L H (26).

Transient Nature of the Low-Dose Signal Initial experiments as diagrammed in Fig. 1 included an induction step at 37 ° for 15 rain. Subsequent experiments showed that this induction step was temperature dependent since signaled cells did not become susceptible to anti-Ig and C if the 37 ° incubation step was replaced by additional incubation at 4°; they did become susceptible if incubated at 30 ° as seen in Fig. 4 (representative data from one of four experiments). In all four experiments induced susceptibility occurred within 10 to 15 rain at 30 ° or 37 ° after which the susceptibility of the cells returned to control levels. The temperature dependence of the process suggests that induction requires active metabolism. DISCUSSION These experiments show that low doses of K L H induce a transient change in K L H helper cells rendering them susceptible to anti-lg and C inactivation. There are several interpretations to our observation. 1) Since the cells are rendered susceptible to anti-Ig and C treatment after antigen induction, unusually low doses of antigen may serve to signal the appear-

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Fla. 4. Transient nattlre and temperature dependence of the low dose signal. KLH-primed spleen cells were signaled with 10-° vg K L H and incubated at 30 ° for various times prior to treatment with anti-Ig and C (direct technique). Control cells were signaled with 10-°/xg K L H but incubated at 4 ° prior to anti-Ig and C treatment. Normal spleen cells were used to replenish B cells. Cells were then cultured with 0,02 ~g T N P - K L H . Each point represents a pool of eight replicate microcultures. Representative data from one of four experiments are

shown.

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ance of Ig-like molecules on helper T cells. Such induced molecules could be intrinsic to the T cell (27-29) with antigen serving as a signal for their appearance through exposure of buried receptor mo!ecules, rearrangement on the surface or de novo synthesis. Alternately, these molecules could be passively acquired from contaminating B cells (30). 2) The anti-Ig sera may possess some anti-membrane activity and it is this specificity which is induced by signaling with low doses of carrier. The cytotoxic activity of two of the three antisera (one was not tested) was inhibited by absorption with purified mouse IgG and we have obtained similar results with three other anti-Ig and one anti-kappa antiserum (not shown). However, we cannot exclude antimembrane activity especially in view of the report that T cells bear a non-Ig antigen that cross reacts with a portion of the kappa chain molecule (31). 3) Rather than inducing the appearance of new molecules, signaling with low doses of K L H could serve to alter the susceptibility of K L H helper cells to C mediated lysis. Alterations in susceptibility to C mediated lysis occur during the cell cycle as shown by Kerbel and Doenhoff (32) who reported that although mitotic 13 cells bear large amounts of Ig they are resistant to lysis by cytotoxic anti-Ig and C whereas non-mitotic B cells are susceptible to lysis. The important point from these experiments is that K L H specific helper cells only become sensitive to the anti-Ig and C reagents after brief exposure to picogram quantities of K L t t . Among the interpretations discussed above we favor the suggestion that low doses of K L H signal the transient appearance of Ig-like molecules on the cell's surface. These molecules, functioning either as antigen receptors or as other regulatory receptors (33) could serve as one of the control mechanisms for engaging helper cells in the immune response. Thus an initial encounter with low doses of antigen serves to signal the transient appearance of Ig-like molecules on the cell's surface. Once armed in this manner, the cell would be prepared for a full antigenic challenge and subsequent participation in the interactions required for triggering 13 cells. However, the transitory nature of the armed state would permit the cell to revert to quiescence without requiring further expenditure of metabolic energy if the full antigenic challenge failed to materialize. Thus the process may be viewed as a means of distinguishing between noise and true antigenic insult at a minimum metabolic price. ACKNOWLEDGMENTS We thank Kathie Pratt for excellent technical assistance and Dr. George Fegan for helpful discussion and advice on the statistical analysis. REFERENCES 1. Paul, W. E., In "Immune Recognition" (A. S. Rosenthal, Ed), Academic Press, New York, 1975. 2. Gershon, R., Contem. Topics [rn~unobiol. 3, 1, 1974. 3. Schimpl, E., and Wecker, E., Transpl. Rev. 23, 176, 1975. 4. Sela, ~I., and Mozes, E., Transpl. Rev. 23, 189, 1975. 5. Feldmann, M., Howard, J., and Desaymard, C., Transpl. Rev. 23, 78, 1975. 6. Howard, J., and Mitchison, N. A., Propr. A~lergy 18, 43, 1975. 7. Bretseher, P., and Cohn, M., Science 169, 1042, 1970. 8. Coutinho, A., and M611er, G., Adv. Im~nunol. 21, 113, 1975. 9. Janeway, C. A., Jr., Transpl. Rev. 29, 164, 1976.

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10. Baltz, M., and Rittenberg, M. B., Fed. Proceed. 33, 765, 1974. 11. Campbell, D., Garvey, J., Cremer, N., and Sussdorf, D., "Methods in Immunology" (2nd edition), W. A. Benjamin, Inc., New York, 1975. 12. Rittenberg, M. B., and Amkraut, A., J. Immunol. 97, 421, 1966. 13. Gallily, R., and Garvey, J. S., Y. Immunol. 1,01, 924, 1968. 14. Rittenberg, M. B., and Pratt, K., Proc. Soc. Exp. Biol. Med. 132, 575, 1969. 15. Kappler, J., J. Immunol. 112, 1271, 1974. 16. Mishell, R., and Dutton, F., J. Exp. Med. 126, 423, 1967. 17. Click, R., Benck, L., and Alter, B., Cell. Immunol. 3, 155, 1972. 18. Cunningham, A., and Szenberg, A., [mmunol. 14, 599, 1968. 19. Kabat, E., and Mayer, M., "Experimental Immunochemistry" (2nd Edition), Charles C Thomas, Springfield, Illinois, 1963. 20. Cohen, A., and Schlesinger, M., Transpl. 10, 130, 1970. 21. Golub, E., Cell. Immunol. 2, 353, 1971. 22. Jennings, I., Baltz, M., and Rittenberg, M. B., Y. Immunol. 115, 1432, 1975. 23. Chan, E., Mishell, R., and Mitchell, G., Science 170, 1215, 1970. 24. Sokal, R. R., and Rholf, F. J., "Biometry," W. H. Freeman and Co., San Francisco, 1969. 25. Dresser, D., and Wortis, H., Nature 2,08, 859, 1965. 26. Feldmann, M., Erb, P., and Kontiainen, S., In "Lymphocytes and Their Interactions" (R. C. Williams, Jr., Ed.), Raven Press, New York, 1975. 27. Roelants, G., Ryden, A., Hagg, L., and Loor, F., Natnre 247, 106, 1974. 28. Binz, H., and Wigzell, H., J. Exp. Med. 14:2, 197, 1975. 29. Eichmann, K., and Rajewsky, K., tT.ur. Y. Immunol. 5, 661, 1975. 30. Hudson, L., Sprent, J., Miller, J., and Playfair, J., Nature 251, 60, 1974. 31. Gottlieb, A. B., Engelhard, M., and Kunkel, H. G., Abstracts of papers presented at the XLI Cold Spring Harbor Symposium on Quantitative Biology Origins of Lymphocyte Diversity. June 1-8 1976. page 5. 32. Kerbel, R., and Doenhoff, M. Nature 250, 342, 1974. 33. M611er, G. (Ed.), Transpl. Rev. 24, 1975.